Pathogenic bacteria require iron for their survival and ability to cause infection. Many bacterial pathogens have evolved sophisticated systems to utilize heme as a primary source of iron. The opportunistic pathogen P. aeruginosa is an increasingly common cause of nosocomial infections with high morbidity associated with such infections. In addition to their natural resistance to many antibiotics the alarming increase in such infections highlights the need for alternate antimicrobial strategies. In previous biochemical and biophysical studies we have characterized the intracellular heme trafficking protein PhuS and the iron-regulated heme oxygenase HemO and have further shown they are critical for heme utilization and virulence. The specific aims of the proposal are to further elucidate the mechanism of extracellular heme utilization and regulation as it relates to virulence. This will be achieved through; i) Elucidating the intracellular heme trafficking pathway and defining the role of biliverdin IX-delta and -beta isomers in the extra-cellular feedback regulation of heme uptake through genetic, biochemical, and metabolomics approaches. ii) Defining the mechanism of heme transfer from PhuS to HemO through a combination of site-directed mutagenesis, biochemical and biophysical studies and iii) Determining the role of the regulatory small RNA PrrH in heme uptake and utilization. These studies will provide new insights into heme and iron homeostasis in bacterial pathogens. We will through this proposal identify new heme metabolic and regulatory pathways as novel antimicrobial targets and advance the current understanding of heme transfer mechanisms.